Interleukin-6, also known as interferon-.beta.2 (IFN-.beta.2), B-cell stimulating factor, hepatocyte stimulating factor and hybridoma growth factor, is a multifunctional cytokine secreted by a variety of cells including monocytes, leukocytes, hepatocytes, fibroblasts, epithelial cells, endothelial cells, glial cells, cardiac myxoma tissue and some bladder carcinomas and cervical cancer cells. Interleukin-6 (hereinafter also referred to as IL-6) regulates the growth and differentiation of many of these cells and appears to play an important role in mediating response to viral and bacterial infections and to shock. Thus, IL-6 has the potential to become a clinically significant compound having multiple therapeutic indications including the treatment of bacterial infection, viral infection and inflammation, and application as an antitumour agent.
The effective clinical use of IL-6 is dependent on, among other things, the provision of IL-6 in a form of pharmaceutically acceptable purity. The purity of a compound for use in a pharmaceutical composition is known to affect the level of its biological activity. Accordingly, as the purity of a compound approaches 100%, its level of biological activity per unit dose is maximized and the compound becomes more pharmaceutically effective. Purity may also impact on the incidence of undesirable side effects associated with the administration of a pharmaceutical compound. Often the identity of impurities which contaminate a compound are unknown and, as a result, the effects upon administration cannot be predetermined. The provision of a pharmaceutical compound in essentially pure form will circumvent the occurrence of complications that may arise from impurities.
One method commonly used to purify proteins is reversed phase high performance liquid chromatography (RP-HPLC). In general, liquid chromatography exploits the variable rates at which proteins migrate in a two phase liquid solvent system through a column typically consisting of silica gel particles. In reversed phase HPLC, a polar mobile phase and a non-polar stationary phase solvent system is utilized to effect separation and thus purification of a protein sample. Typically, the mobile phase consists of water and an organic solvent combined with an ion-pairing agent or charge modifier. The relative proportions of the water and organic solvent in the mobile phase may be altered gradiently over the course of the protein migration through the silica column in order to augment the analysis. A protein preparation eliciting a single peak when analyzed using liquid chromatography (as identified by UV absorbance at a wavelength of 214 nm or 280 nm) was heretofore believed to consist of a single protein species and has previously been characterized as being essentially pure.
However, the sensitivity of such HPLC techniques has been found to be secondary to the sensitivity of the method of capillary electrophoresis in analyzing the purity of a protein sample. Capillary electrophoresis separates proteins based on their mass/charge ratio within a capillary having a bore of miniscule diameter, as described by Gordon et al. in Science, 1988, 242:224. Briefly, aqueous samples of a protein preparation to be analyzed are drawn by vacuum into the capillary and subjected to an electric field. Migration of the protein species through the capillary is monitored by detecting UV absorbance, usually at 214 nm. Protein samples determined to be essentially pure by HPLC have subsequently been analyzed by capillary electrophoresis, the results of which reveal that, in fact, the sample contains more than one compound as indicated by the occurrence of more than one absorption peak, and thus is not essentially pure as originally determined.
Reversed phase HPLC has been used previously as a step in the purification of IL-6. Van Damme et al. (Eur. J. Biochem, 1987, 168:543) describe a 5-step purification process for IL-6 produced from human fibroblast cultures. The purification steps included in the process were silicic acid adsorption, antibody affinity chromatography, gel filtration chromatography, cation-exchange chromatography and reverse-phase HPLC. The process was described as yielding pure IL-6 as determined using the method of gel electrophoresis, i.e. approximately pure IL-6.
Methods for making and purifying recombinantly-produced interleukin-6 have also been described in the art. For example, recombinantly produced murine IL-6 has been purified by ultrafiltration followed by fractionation using reverse phase liquid chromatography initially under acidic conditions and then under alkaline conditions as described by Lee et al. (Ann. NY Acad. Sci. 1989, 557:215). A further method of providing recombinant IL-6 has been described by Clark et al. in WO 88/00206. In particular, this reference teaches a method for the bacterial production of non-glycosylated IL-6, a form of IL-6 which does not naturally exist in humans. In both cases IL-6 is provided, as in the process of Van Damme, supra, in a form estimated to be pure by gel electrophoresis.
To date, essentially pure human interleukin-6, as indicated by single peak absorption using capillary electrophoresis, and a method for obtaining such essentially pure human interleukin-6 has not been reported.
Thus, an object of the present invention is to provide human IL-6 in essentially pure form, free from contaminants detectable by capillary electrophoresis (CE). A further object of the present invention is to provide a method to obtain such essentially pure human IL-6.